GSM7119479: KD, replicate 3, snRNAseq; Mus musculus; RNA-Seq
Kidney cortex was dissected out after PBS washout via aortic perfusion and snap-frozen in liquid nitrogen immediately after dissection. Frozen tissue was stored at -80 °C until subsequent tissue processing. Prepare the nuclei isolation buffer (NIB, includes 146 mM NaCl, 10 mM Tris-HCl, 1 mM CaCl2, 21 mM MgCl2, 0.03% Tween-20, 0.01% BSA, pH 7.5), and add 1 tablet of cOmplete ULTRA per 10 ml NIB. The samples were ground for 30 times in 2ml NIB1 (4mL NIB + 20µl RNasin Plus + 20µl SUPERaseIN) with a 2ml Dounce grinder and a loose pestle, then homogenate was passed through a 200 µm strainer. The homogenate was ground for 15 times with a tight pestle, then 2 ml NIB1 was added and the homogenate was incubated for 5 min on ice. The homogenate was passed through a 40 µm strainer, then centrifuged at 500g for 5 min at 4°C. The pellet was resuspended in 4 ml NIB2 (4mL NIB + 4µl RNasin Plus + 4µl SUPERaseIN) and the suspension was incubated on ice for 5 min, then centrifuged at 500g for 5 min at 4°C. The pellet was resuspended in 1.5 ml of nuclei resuspension buffer (NSB, 10mL DPBS + 10µl RNasin Plus), then centrifuged at 500g for 5 min at 4°C. Repeat this step, then the suspension was passed through a 5 µm strainer. The suspension was centrifuged at 500g for 5 min at 4°C, and the pellet was resuspended in 10 ml NSB. The nuclei suspension was mixed with 5µl Vybrant Ruby stain and incubated on ice for 15 min. The nuclei were sorted in 500 µl final resuspension buffer (FSB, 1ml DPBS with 1% BSA + 5 µl Protector RNase inhibitor) with a low flow rate and pressure to ensure high viability. Two main gates were used. A 561+683 emission for the ruby stain and a 488 530/40 emission for GFP. Low trigger pulse width was used as singlet discriminator. One hundred thousand nuclei were collected and centrifuged at 500g for 5 min at 4°C. The top supernatant was carefully removed, and 50 µl volume was left to resuspend the nuclei pellet. Single nucleus RNA-seq was performed using a Chromium Next GEM Single Cell 3′ Reagent Kit v3.1 (10x Genomics). Single nuclei were partitioned in droplets with single Gel Beads, which contained primers with cell-tagging indexes. Single nucleus suspensions with concentration of at least 300/µl were loaded targeting 10,000 nuclei per sample. The resulting cDNA was profiled on a Bioanalyzer NanoChip (Agilent), then used as a template for library preparation according to the reagent kit protocol. ( https://www.10xgenomics.com/support/single-cell-gene-expression/documentation/steps/library-prep/chromium-single-cell-3-reagent-kits-user-guide-v-3-1-chemistry )